US7575910B2 - Method for producing L-fuculose and method for producing L-fucose - Google Patents

Method for producing L-fuculose and method for producing L-fucose Download PDF

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US7575910B2
US7575910B2 US11/532,618 US53261806A US7575910B2 US 7575910 B2 US7575910 B2 US 7575910B2 US 53261806 A US53261806 A US 53261806A US 7575910 B2 US7575910 B2 US 7575910B2
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fuculose
fucitol
protein
fucose
activity
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US20070026504A1 (en
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Shunichi Suzuki
Kunihiko Watanabe
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Ajinomoto Co Inc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/02—Monosaccharides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
    • C12N9/0036—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on NADH or NADPH (1.6)

Definitions

  • the present invention relates to simple and efficient methods for producing L-fuculose and L-fucose.
  • L-Fucose (L-6-deoxygalactose) is ubiquitous throughout the living world, mostly as a non-reducing terminal sugar of various carbohydrates.
  • fucoidan in seaweed is a polysaccharide which contains L-fucose.
  • L-Fucose is believed to be involved in the uptake of serum glycoprotein into the liver, and is able to induce receptors for macrophage migration inhibitory factor in vivo.
  • L-Fucose has also been studied relative to its association with various diseases. For example, L-fucose has been studied as a pharmaceutical intermediate.
  • L-fuculose-1-phosphate As a synthetic method using an enzyme, converting L-fuculose-1-phosphate into L-fuculose using acid phosphatase and further converting into L-fucose using L-fucose isomerase has been described (International Publication WO97/15683 Pamphlet).
  • L-fuculose-1-phosphate is expensive. Therefore, there is a demand in the art for a method which enables production at lower cost.
  • the acetobacterium has been extensively studied in relation to the enzyme acting on a sugar as a substrate (e.g., JP H8-242850 A, Canadian Journal of Chemistry 45: 741-744 (1967), Biosci. Biotechnol. Biochem. 65: 2755-2762 (2001)).
  • a protein having a fucitol dehydrogenase activity was derived from a microorganism. Furthermore, techniques for synthesizing L-fuculose from L-fucitol when there is no protein which is able to synthesize a ketohexose other than L-fuculose from L-fucitol, or the activity thereof is inhibited.
  • An industrially suitable method for producing L-fuculose, which is an intermediate in the production of L-fucose, is described, as well as an industrially advantageous method for producing L-fucose. That is, aspects of the present invention include the following.
  • One aspect of the present invention includes a method for producing L-fuculose comprising contacting L-fucitol with a protein derived from a microorganism, the protein having a dehydrogenase activity which results in the synthesis of L-fuculose from L-fucitol.
  • Another aspect includes a method for producing L-fuculose as described above, wherein the protein is isolated from an acetobacterium.
  • Another aspect includes a method for producing L-fuculose comprising contacting L-fucitol with a composition selected from the group consisting of a microorganism which is able to synthesize L-fuculose from L-fucitol so that the amount of L-fuculose is 50% by weight or more of total L-fucitol oxide, a culture of the microorganism, a treated microbial cell product of the microorganism, and combinations thereof.
  • Another aspect includes a method for producing L-fuculose comprising contacting L-fucitol with a composition selected from the group consisting of at least one microorganism of Gluconobacter xylinus subsp. xylinus and Gluconobacter oxydans , a culture of the microorganism, a treated microbial cell product of the microorganism, and combinations thereof.
  • a composition selected from the group consisting of at least one microorganism of Gluconobacter xylinus subsp. xylinus and Gluconobacter oxydans , a culture of the microorganism, a treated microbial cell product of the microorganism, and combinations thereof.
  • Another aspect includes a method for producing L-fuculose comprising contacting L-fucitol with a composition selected from the group consisting of a microorganism having a protein having a dehydrogenase activity which results in the synthesis of L-fuculose from L-fucitol, the microorganism not having a substantial ability to synthesize a ketohexose, other than L-fuculose, from L-fucitol, a culture of the non-genetically engineered microorganism, a treated microbial cell product of the non-genetically engineered microorganism, and combinations thereof.
  • Another aspect includes producing L-fuculose as described above wherein a gene has been knocked out in said microorganism which encodes a protein having an activity which results in the synthesis of a ketohexose, other than L-fuculose, from L-fucitol.
  • Another aspect includes producing L-fuculose as described above, wherein the microorganism has been transformed to be capable of expressing said protein having a dehydrogenase activity.
  • Another aspect includes producing L-fuculose as described above, wherein a gene has been knocked out in said microorganism which encodes a protein having an activity which results in the synthesis of a ketohexose, other than L-fuculose, from L-fucitol.
  • Another aspect includes producing L-fuculose as described above, wherein L-fuculose is synthesized from L-fucitol when synthesis of a ketohexose, other than L-fuculose, from L-fucitol is inhibited.
  • Another aspect includes producing L-fuculose as described above, wherein L-fuculose is synthesized from L-fucitol at a pH which inhibits synthesis of a ketohexose, other than L-fuculose, from L-fucitol.
  • Another aspect includes producing L-fuculose as described above, wherein L-fuculose is synthesized from L-fucitol in the presence of a bivalent ion chelator when synthesis of a ketohexose, other than L-fuculose, from L-fucitol is inhibited.
  • Another aspect includes producing L-fuculose as described above, wherein L-fuculose is synthesized from L-fucitol in the presence of a protein having an activity to produce NAD from NADH.
  • Another aspect includes producing L-fucose comprising synthesizing L-fuculose by contacting L-fucitol with a protein derived from a microorganism having a dehydrogenase activity which results in the synthesis of L-fuculose from L-fucitol, and synthesizing L-fucose by contacting L-fuculose with a protein having an activity to synthesize L-fucose from L-fuculose.
  • Another aspect includes producing L-fucose comprising synthesizing L-fuculose by contacting L-fucitol with a composition selected from the group consisting of a microorganism having an ability to synthesize L-fuculose from L-fucitol so that the amount of L-fuculose is 50% by weight or more of total L-fucitol oxide, a culture of the microorganism, a treated microbial cell product of the microorganism, and combinations thereof, and synthesizing L-fucose by contacting L-fuculose with a protein having an activity to synthesize L-fucose from L-fuculose.
  • Another aspect includes producing L-fucose comprising synthesizing L-fuculose by contacting L-fucitol with a composition selected from the group consisting of a microorganism having a protein having a dehydrogenase activity which results in the synthesis of L-fuculose from L-fucitol, wherein the microorganism does not have a substantial ability to synthesize a ketohexose, other than L-fuculose, from L-fucitol, a culture of the microorganism, a treated microbial cell product of the microorganism, and combinations thereof, and synthesizing L-fucose by contacting L-fuculose with a protein having an activity to synthesize L-fucose from L-fuculose.
  • Another aspect includes producing L-fucose as described above, wherein a gene has been knocked out in said microorganism, wherein said gene encodes a protein having an activity to synthesize a ketohexose, other than L-fuculose, from L-fucitol.
  • Another aspect includes producing L-fucose as described above, wherein said microorganism has been transformed to be capable of expressing a protein having a dehydrogenase activity which results in the synthesis of L-fuculose from L-fucitol.
  • Another aspect includes producing L-fucose as described above, wherein said microorganism expresses a protein having an activity to synthesize L-fucose from L-fuculose.
  • Yet another aspect of the present invention includes a protein selected from the group consisting of: (A) a protein comprising the amino acid sequence of SEQ ID NO:16, and (B) a protein comprising the amino acid sequence of SEQ ID NO.16, wherein said sequence includes one or several amino acid mutations selected from the group consisting of substitution, deletion, insertion, addition, and inversion, and having a dehydrogenase activity which results in the synthesis L-fuculose from L-fucitol.
  • Another aspect includes a polynucleotide encoding the protein as described above.
  • Another aspect includes a polynucleotide selected from the group consisting of: (A) a polynucleotide comprising the nucleotide sequence according to SEQ ID NO:15, and (B) a polynucleotide which hybridizes with a polynucleotide comprising a nucleotide sequence which is complementary to the nucleotide sequence of SEQ ID NO:15 under stringent conditions and encodes a protein having a dehydrogenase activity which results in the synthesis of L-fuculose from L-fucitol.
  • Another aspect includes a protein selected from the group consisting of: (A) a protein comprising the amino acid sequence of SEQ ID NO:18, and (B) a protein comprising the amino acid sequence of SEQ ID NO: 18, wherein said sequence includes one or several amino acid mutations selected from the group consisting of substitution, deletion, insertion, addition, and inversion, and having an NADH oxidase activity.
  • Another aspect includes a polynucleotide encoding the protein as described above.
  • Another aspect includes a polynucleotide selected from the group consisting of: (A) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:17, and (B) a polynucleotide which hybridizes with a polynucleotide comprising a nucleotide sequence which is complementary to the nucleotide sequence of SEQ ID NO:17 under stringent conditions and encodes a protein having an activity to produce NAD from NADH.
  • Another aspect includes a recombinant polynucleotide comprising the polynucleotide described in paragraph [0032] incorporated therein.
  • Another aspect includes a recombinant polynucleotide comprising the polynucleotide described in paragraph [0035] incorporated therein.
  • Another aspect includes a recombinant polynucleotide comprising the polynucleotide described in paragraph [0032] and the polynucleotide described in paragraph incorporated therein.
  • Another aspect includes a transformant comprising the recombinant polynucleotide described in paragraph [0037], the transformant being capable of expressing a protein having a dehydrogenase activity which results in the synthesis L-fuculose from L-fucitol.
  • Another aspect includes a method for producing a protein comprising culturing the transformant described in paragraph [0040], wherein the transformant is a microorganism, in a medium wherein said protein accumulates in the medium and/or in the microorganism.
  • Yet another aspect includes a method for producing L-fuculose comprising adding to a reaction system containing L-fucitol a composition selected from the group consisting of the transformant described in paragraph [0040], wherein the transformant is a microorganism, a culture of the microorganism, a treated microbial cell product of the microorganism, and combinations thereof, wherein L-fuculose is synthesized from L-fucitol.
  • Another aspect includes a method for producing L-fucose comprising synthesizing L-fuculose by adding to a reaction system containing L-fucitol a composition selected from the group consisting of the transformant as described in paragraph [0040], wherein the transformant is a microorganism, a culture of the microorganism, a treated microbial cell product of the microorganism, and combinations thereof, wherein L-fuculose is synthesized from L-fucitol, and synthesizing L-fucose by contacting L-fuculose with a protein having an activity to synthesize L-fucose from L-fuculose.
  • Another aspect includes a transformant comprising the recombinant polynucleotide described in paragraph [0037] and the recombinant polynucleotide described in paragraph [0038] incorporated therein, the microorganism being capable of expressing a protein having a dehydrogenase activity which results in the synthesis of L-fuculose from L-fucitol and a protein having an activity to produce NAD from NADH.
  • Another aspect includes a method for producing a protein comprising culturing a microorganism which has been transformed with the recombinant polynucleotide described in paragraph [0038] in a medium, wherein said microorganism is capable of expressing said protein in the medium and/or in the microorganism.
  • Another aspect includes a method for producing L-fuculose comprising adding to a reaction system containing L-fucitol a composition selected from the group consisting of the transformant described in paragraph [0044], wherein the transformant is a microorganism, a culture of the microorganism, a treated microbial cell product of the microorganism, and combinations thereof, wherein L-fucolose is synthesized from L-fucitol.
  • Another aspect includes a method for producing L-fucose comprising synthesizing L-fuculose wherein one or more selected from the group consisting of the transformant described in paragraph [0044], wherein the transformant is a microorganism, a culture of the microorganism, a treated microbial cell product of the microorganism, and combinations thereof, are added to a reaction system containing L-fucitol, wherein L-fuculose is synthesized from L-fucitol, and synthesizing L-fucose by contacting L-fuculose with a protein having an activity to synthesize L-fucose from L-fuculose.
  • Another aspect includes a transformant comprising the recombinant polynucleotide described in paragraph [0039], the transformant being capable of expressing a protein having a dehydrogenase activity which results in the synthesis of L-fuculose from L-fucitol and a protein having an activity to produce NAD from NADH.
  • Another aspect includes a method for producing a protein having a degydrogenase activity which results in the synthesis of L-fuculose from L-fucitol and a protein having an activity to produce NAD from NADH comprising culturing the transformant described in paragraph [0048] in a medium, wherein the transformant is a microorganism, and wherein said proteins accumulate in the medium and/or in the microorganism.
  • Another aspect includes a method for producing L-fuculose comprising adding a composition selected from the group consisting of the transformant described in paragraph [0048], wherein the transformant is a microorganism, a culture of the microorganism, a treated microbial cell product of the microorganism, and combinations thereof, to a reaction system containing L-fucitol, wherein L-fuculose is synthesized from L-fucitol.
  • Another aspect includes a method for producing L-fucose comprising synthesizing L-fuculose wherein a composition selected from the group consisting of the transformant described in paragraph [0048], wherein the transformant is a microorganism, a culture of the microorganism, a treated microbial cell product of the microorganism, and combinations thereof are added to a reaction system containing L-fucitol, wherein L-fuculose is synthesized from L-fucitol, synthesizing L-fucose by contacting L-fuculose with a protein having an activity to synthesize L-fucose from L-fuculose.
  • FIG. 1 shows the reaction process from D-galactose to L-fucose.
  • FIG. 2 shows the amount of L-fucitol converted by FucI-expressing acetobacterium.
  • FIG. 3 shows the amounts of byproducts (BP) which are produced during the production of L-fuculose from L-fucitol under different pH conditions.
  • FIG. 4 shows the amounts of byproducts (BP) which are produced when L-fuculose is produced from L-fucitol in the presence of EDTA.
  • FIG. 5 shows the amounts of byproducts (BP) which are produced when L-fuculose is produced from L-fucitol using a knock-out sidA gene.
  • FIG. 6 shows an SDS-PAGE profile of purified FcDH.
  • FIG. 7 shows an SDS-PAGE profile of purified NOX.
  • FIG. 8 shows the optimal pH when purifying FcDH.
  • FIG. 9 shows the pH stability of purified FcDH.
  • FIG. 10 shows the optimal temperature when purifying FcDH.
  • FIG. 11 shows the temperature stability of purified FcDH.
  • FIG. 12 shows the specific activity of purified FcDH at various concentrations of D-arabitol (A) and L-fucitol (B).
  • FIG. 13 shows the FcDH-oxidized products of D-arabitol, D-mannitol and D-sorbitol, as measured by HPLC analysis.
  • FIG. 14 shows the specific activity of purified FcDH at various NAD concentrations.
  • FIG. 15 shows the optimal pH when purifying NOX.
  • FIG. 16 shows the pH stability of purified NOX.
  • FIG. 17 shows the optimal temperature when purifying NOX.
  • FIG. 18 shows the temperature stability of purified NOX.
  • FIG. 19 shows the specific activity of purified NOX at various concentrations of flavin coenzymes (FAD, riboflavin and FMN).
  • FIG. 20 shows the specific activity of purified NOX at various concentrations of NADH ( FIG. 20 (A)).
  • FIG. 20 (B) shows Lineweaver-Burk plot of (A).
  • FIG. 21 shows the SDS-PAGE profiles of cell free extracts prepared from E. coli /pUC18 , E. coli /pIEX11 , E. coli /pFEX3052, E. coli /pFNEX4105 and E. coli /pFNIEX5706.
  • FIG. 22 shows the SDS-PAGE profile of rFcDH purified from E. coli /pFEX3052.
  • FIG. 23 shows the SDS-PAGE profile of rNOX purified from E. coli /pFNEX4105.
  • FIG. 24 shows the time courses of L-fucitol conversion using FcDH and NOX purified from G. oxydans.
  • FIG. 25 shows the time courses of L-fucitol conversion using rFcDH and rNOX prepared from recombinant E. coli.
  • FIG. 26 shows the time courses of L-fucitol conversion by recombinant E. coli intact cells.
  • FIG. 27 shows the time courses of L-fucitol conversion in the presence of catalase.
  • L-fuculose is synthesized from L-fucitol using a protein derived from a microorganism.
  • This protein has dehydrogenase activity, which enables synthesis of L-fuculose from L-fucitol.
  • a protein is described which is able to synthesize L-fuculose from L-fucitol independently from the protein which synthesizes a ketohexose, other than L-fuculose, from l-fucitol in the microorganism. Therefore, it is possible to reduce or eliminate the byproduct production.
  • the protein is derived from a microorganism, isolation and purification of the protein are easier than when purifying from plants and animals. Another advantage is that the protein will be compatible with a host when expressed on a large scale in the microorganism. Cultivation of a microorganism is easier than cultivation of plant or animal cells, and therefore it is easy to obtain a sufficient amount for industrial production.
  • amino acid and nucleotide sequence of this protein was determined based on isolationg of the protein from Gluconobacter oxydans.
  • the method for producing L-fuculose may reduce the cost since it uses the inexpensive starting material L-galactose, and is thus advantageous for industrial production because L-fucitol is easily obtainable from L-galactose.
  • the method for obtaining L-fucitol from D-galactose is described in, for example, Carbohydrate research 270; 93-96 (1995).
  • the protein is derived or isolated from a microorganism, and has dehydrogenase activity, which allows it to synthesize L-fuculose from L-fucitol.
  • the microorganisms which may be used include prokaryotic and eukaryotic microorganisms, and viruses, but exclude plant cells including algae. Examples of the microorganism may include those belonging to an acetobacterium. Acetobacterium may specifically include bacteria belonging to genera Gluconobacter and Acetobacter , and more specifically bacteria belonging to Gluconobacter oxydans, Gluconobacter frateurii, Acetobacter turbidans, Gluconobacter roseus, Gluconobacter cerinus, Gluconobacter oxydans subsp.
  • the protein may be obtained from the various bacterial strains described in Table 1.
  • the microorganism may be cultured in an appropriate medium, depending on its type.
  • the medium is not particularly limited as long as the microorganism can grow in the medium, and may be any of the standard media which typically contains sources of carbon, nitrogen, phosphorous, sulfur, inorganic ions, and further optionally containing organic nutrients.
  • Any carbon source which the microorganism can utilize may be used. Specifically, saccharides such as glucose, fructose, maltose, and amylose, alcohols such as sorbitol, ethanol, and glycerol, organic acids such as fumaric acid, citric acid, acetic acid, and propionic acid and salts thereof, hydrocarbons such as paraffin, and mixtures thereof, may be used.
  • saccharides such as glucose, fructose, maltose, and amylose
  • alcohols such as sorbitol, ethanol, and glycerol
  • organic acids such as fumaric acid, citric acid, acetic acid, and propionic acid and salts thereof
  • hydrocarbons such as paraffin, and mixtures thereof
  • ammonium salts of inorganic acids such as ammonium sulfate and ammonium chloride
  • ammonium salts of organic acids such as ammonium fumarate and ammonium citrate
  • nitrate salts such as sodium nitrate and potassium nitrate
  • organic nitrogen compounds such as peptone, yeast extract, meat extract and corn steep liquor, or mixtures thereof, may be used.
  • inorganic salts, trace metal salts and vitamins found in standard media may be used as the nutrient source, by appropriately mixing them.
  • Conditions for cultivation are not particularly limited.
  • the cultivation may be performed under aerobic conditions, while appropriately controlling pH and temperature so that they range from pH 5 to 8 and from 15 to 40° C. for about 12 to 70 hours.
  • Microbial cell extracts are typically the starting material which is subjected to purification. If non-disrupted or non-lysed cells remain, further precipitated residues can be removed by subjecting the solubilized solution to re-centrifugation.
  • L-fucitol may be placed in contact with the protein in a medium.
  • L-fucitol and the protein may be added to a buffer and allowed to react.
  • the ranges of pH, temperature, reaction time, and amount of the added enzyme may be appropriately adjusted depending on the type of the protein.
  • the pH is preferably 5 to 11, and more preferably 7 to 10.
  • the temperature is preferably 20 to 50° C. and more preferably 25 to 40° C.
  • a method of using a microorganism, a culture of the microorganism, or a treated microbial cell product of the microorganism will now be described.
  • Another embodiment for the method of synthesizing L-fuculose using the aforementioned protein is to use a microorganism which contains the aforementioned protein, a culture thereof, or a treated microbial cell product thereof.
  • the microorganism may also synthesize a ketohexose other than L-fuculose (hereinbelow referred to as a byproduct).
  • a byproduct a ketohexose other than L-fuculose
  • a microorganism which satisfies at least two criteria: (i) the ability to synthesize L-fuculose from L-fucitol, and (ii) the ability to synthesize L-fuculose to such an extent that the amount of L-fuculose thus produced accounts for preferably 50% or more, more preferably 70% or more, and still more preferably 90% or more of total L-fucitol oxide generated by oxidizing L-fucitol.
  • the microorganism which satisfies both the aforementioned criteria may include Gluconobacter xylinus subsp. xylinus and Gluconobacter oxydans , more preferably Gluconobacter xylinus subsp. xylinus ATCC 53582 strain and ATCC 23767 strain, and Gluconobacter oxydans IFO 3189 strain, and still more preferably Gluconobacter xylinus subsp. xylinus ATCC 53582 strain. These microorganisms do not synthesize substantial amounts of ketohexoses other than L-fuculose, or if so, the amounts are very small.
  • a preferable embodiment of the present invention is a method of using a non-genetically engineered microorganism which contains the protein having the dehydrogenase activity which results in synthesis of 1-fuculose from L-fucitol, wherein the microorganism does not have a subtantial ability to synthesize ketohexoses, other than L-fuculose, from L-fucitol.
  • the phrase “does not have a substantial ability” means that the amount of the ketohexose, other than L-fuculose, byproducts is not more than the detection limit under the conditions shown in Example 1.
  • the phrase “does not have a substantial ability” means that the amount of the non-L-fuculose ketohexose byproducts is about 1 mM or less.
  • a “genetically engineered microorganism” refers to a microorganism in which a gene has been artificially engineered using genetic engineering techniques, and does not include naturally occurring mutants.
  • the phrase “non-genetically engineered microorganism” refers to microorganisms which are not genetically engineered.
  • culture of the microorganism means the culture obtained after culturing the microorganism.
  • the culture of the microorganism may specifically contain microbial cells of the microorganism, the medium used for the cultivation of the microorganism, and/or a mixture of substances produced by the microorganism, and the supernatant thereof.
  • treated microbial cell product refers to the product obtained after some artificial manipulation of the microbial cells.
  • the treated microbial cell product may contain, for example, disrupted microbial cells, lysed cells, and lyophilized cells.
  • the treated microbial cell product may also be a crude protein collected by treating the microbial cell, or a purified protein.
  • the purified protein may be a partially purified protein obtained by various purification methods, and an immobilized protein obtained by immobilizing the protein via covalent bonds, an absorption method, or an inclusion method.
  • the treated microbial cell product may contain matter that includes the content of the cells of the microorganism. Therefore, when the treated microbial cell product is used, proteins responsible for synthesizing the byproduct may be present. Thus, it is preferable to use a microorganism which is able to synthesize L-fuculose so that the total amount of L-fuculose accounts for 50% by weight or more of total L-fucitol oxide.
  • the microorganism may be a source of this protein.
  • the microorganism is not limited by whether the microorganism produces the byproduct or not.
  • Synthesis of L-fuculose using the microorganism may be conducted so that the protein which is able to synthesize L-fuculose may act upon the substrate L-fucitol.
  • the microorganism may be cultured and L-fucitol may be added to the culture.
  • Synthesis with the treated microbial cell product may be performed by mixing the treated microbial cell product with L-fucitol so that the protein which is able to synthesize L-fuculose may act upon the substrate L-fucitol. That is, the reaction system may be constructed in the same manner as the reaction system that uses an enzyme or a bioactive substance.
  • Gene knockout refers to a procedure by which the gene encoding the target protein is modified so to reduce or eliminate the function of the intact protein.
  • the “gene” refers to a molecule which encodes genetic information, and includes DNA, RNA, and polynucleotides such as hybrid molecules thereof or chimera molecules thereof.
  • the gene knockout procedure includes introducing a mutation by deletion, substitution, insertion, and/or inversion so that the protein itself is modified, or inhibiting the expression of the protein by inhibiting transcription or translation.
  • the method for knocking out the gene is not particularly limited.
  • the gene may be mutated or the gene may be deleted.
  • Inactivation of the gene may be performed by general methods such as mutagenesis by UV irradiation or treatment with N-methyl-N′-nitro-N-nitrosoguanidine, site-directed mutagenesis, homologous recombination, or insertion-deletion mutagenesis, often referred to as homologous recombination or “Red-driven integration” (Datsenk K. A. and Wanner B. L., Proc. Natl. Acad. Sci. USA, 97(12:6640-45, 2000).
  • a recombinant DNA construct which contains the the target gene, but a portion of the target gene has been deleted so to inactivate the expression and producution of the dehydrogenase.
  • a microorganism in which the chromosomal dehydrogenase gene has been deleted may be obtained by transforming the microorganism with the above-described DNA construct to cause recombination between the inactivated dehydrogenase gene and the chromosomal peptidase gene.
  • the protein which is able to synthesize a ketohexose other than L-fuculose from L-fucitol may also be subjected to a gene knockout procedure.
  • a method for using the microorganism which is not able to substantially synthesize a ketohexose other than L-fuculose from L-fucitol and wherein said microorganism is transformed so that is expresses the protein which has dehydrogenase activity which allows it to synthesize L-fuculose from L-fucitol.
  • L-fuculose production can be obtained by by modifying the microorganism by introducing the gene which expresses L-fuculose. It is preferable to select a microorganism which is unable to produce the byproduct to facilitate efficient production of L-fuculose without unnecessary byproducts.
  • a microorganism which is not able to to substantially synthesize a ketohexose other than L-fuculose from L-fucitol may be chosen, whether the gene which encodes a protein which is able to synthesize a ketohexose has been knocked out, or whether the microorganism does not contain such an activity in its native state.
  • Examples of the protein to be subjected to gene knockout may include the protein which is able to synthesize D-xylulose from D-arabitol.
  • the transformant may be produced in accordance with standard methods.
  • the transformant may be obtained as follows.
  • a protein which is able to synthesize L-fucose is obtained and purified from the aforementioned microorganism, and its amino acid sequence is determined.
  • the amino acid sequence may be determined using the Edman method (Edman, P., Acta Chem. Scand. 4, 227 (1950)).
  • the amino acid sequence may also be determined using a sequencer (Applied Biosystems).
  • the first 30 amino acid residues from the N terminus is determined, and this sequence is reverse translated to determine the nucleotide sequence of the DNA. Universal codons are employed for deducing the nucleotide sequence of the DNA.
  • a DNA molecule of about 30 base pairs is synthesized.
  • the method for synthesizing the DNA molecule is disclosed in Tetrahedron Letters, 22, 1859 (1981).
  • the DNA molecule may also be synthesized using a synthesizer supplied from Applied Biosystems.
  • the DNA molecule may be utilized as a probe to isolate the full length DNA from a chromosomal gene library from the microorganism.
  • the DNA molecule may also be utilized as a primer in PCR to amplify the.
  • the PCR method is described in White, T. J. et al., Trends Genet. 5, 185 (1989).
  • the method for preparing chromosomal DNA and the method for isolating the object DNA molecule from the gene library using the DNA molecule as the probe are described in Molecular Cloning, 2nd edition, Cold Spring Harbor press (1989).
  • the method for determining the nucleotide sequence of the isolated DNA encoding the protein is described in A Practical Guide to Molecular Cloning, John Wiley & Sons, Inc. (1985).
  • the nucleotide sequence may also be determined using a DNA sequencer (Applied Biosystems).
  • the DNA molecule isolated by the aforementioned method may be introduced into a host. That is, the isolated DNA molecule is incorporated into an expression vector so that it is able to be expressed, and this is introduced into the host cell.
  • bacterial cells When the protein is produced on a large scale using the recombinant gene technology, bacterial cells, actinomycetal cells, yeast cells, fungal cells, plant cells, and animal cells may be used as hosts.
  • enteric bacteria preferably Escherichia coli is suitable, because this bacteria is commonly used for producing proteins on a large scale. The method for producing the protein using transformed Escherichia coli will be described below.
  • Promoters which are conventionally used for the production of a xenogeneic protein in Escherichia coli may be used. Examples thereof may include potent promoters such as T7 promoter, lac promoter, trp promoter, trc promoter, and tac promoter, as well as P R promoter and P L promoter of lambda phage.
  • the promoter is ligated to the gene encoding the protein so to line up the reading frames of the codons.
  • the ligation may be performed at appropriate restriction enzyme sites.
  • a synthetic DNA of an appropriate sequence may be utilized.
  • the terminator may include T7 terminator, fd phage terminator, T4 terminator, a terminator of a tetracycline resistant gene, and a terminator of Escherichia coli trpA gene.
  • the so-called multicopy-type vectors are preferable to introduce the gene into E. coli .
  • Examples thereof may include plasmids which have a replication origin derived from ColE1, e.g., pUC based plasmids, pBR322 based plasmids, or derivatives thereof.
  • the term “derivative” means the plasmid which is modified by substitution, deletion, insertion, addition and/or inversion. The modification may be by mutagenesis with a mutagen or UV irradiation or natural mutation.
  • vectors for example, pUC19, pUC18, pBR322, pHSG299, pHSG298, pHSG399, pHSG398, RSF1010, pMW119, pMW118, pMW219, and pMW218 may be used. Additionally, vectors such as phage DNA or transposon DNA may be used.
  • the vector has a marker such as an ampicillin resistant gene.
  • Expression vectors with the potent promoter are commercially available [pUC series (Takara Shuzo Co., Ltd.), pPROK series (Clonetech), pKK233-2 (Clonetech)].
  • a DNA fragment obtained by ligating the promoter, the gene encoding a peptide synthesizing enzyme, or a fusion protein of the peptide synthesizing enzyme and another protein, and the terminator is ligated to the vector DNA to yield a recombinant DNA.
  • Escherichia coli is then transformed with this recombinant DNA, and cultured to express and produce the protein.
  • the strain which is usually used for the expression of the xenogeneic gene may be used.
  • Escherichia coli JM109 strain which is one of Escherichia coli K12 subspecies is preferable.
  • the method for transforming and the method for selecting the transformant are described in Molecular Cloning, 2nd edition, Cold Spring Harbor press (1989).
  • Media such as M9-casamino acid medium and LB medium usually used for culturing Escherichia coli may be used for the cultivation.
  • the cultivation and production induction conditions are selected depending on the marker and promoter used, and the host microorganism.
  • the protein may be collected by the methods previously described.
  • the microorganism synthesizes a byproduct other than L-fuculose, by oxidizing L-fucitol under conditions which inhibit the production of the byproduct, it is possible to produce L-fuculose in an industrially efficient manner.
  • the pH may be adjusted, or an inhibitor which selectively inhibits the enzyme which controls the production of the byproduct may be added.
  • the pH level which inhibits the production of the byproduct varies depending on the type of the microorganism.
  • a preliminary experiment can be performed depending on the type of the microorganism.
  • the preliminary experiment may be performed under the conditions shown in the following Example, and those skilled in the art may set the pH condition to inhibit the byproduct based on the following example.
  • the inhibitor for the production of the byproduct may be added to the reaction system. It is desirable that the inhibitor does not inhibit the production of L-fuculose from L-fucitol at all, but any inhibitor can be used as long as L-fuculose is sufficiently produced relative to the production of the byproduct.
  • the inhibitor include a bivalent ion chelator and preferably EDTA (ethylenediamine tetraacetate).
  • the microorganism which is able to synthesize L-fuculose from L-fucitol may also have the protein which can produce NAD from NADH in some cases.
  • the microorganism or the treated microbial cell product may be added to the reaction system. If the protein which is able to produce NAD from NADH is not present in the reaction system, such a protein may be added.
  • Many dehydrogenases are able to produce NAD from NADH, and the reduction ability of these dehydrogenases may be utilized. In this case, NADH functions as a coenzyme, and thus, it is necessary to add additional substrate for the reaction.
  • oxidase and peroxidase which oxidize NADH to NAD may be used.
  • the substrate required is oxygen for NADH oxidase or hydrogen peroxide for peroxidase.
  • the substrate is easily supplied into the reaction solution by stirring the reaction solution.
  • hydrogen peroxide is very inexpensive and therefore advantageous in terms of cost.
  • Oxidase and peroxidase are publicly known and commercially available.
  • the method for producing L-fucose of the present invention includes a step of synthesizing L-fuculose, and a step of synthesizing L-fucose from L-fuculose.
  • the aforementioned steps may be performed in separate reaction systems or in a combined reaction system.
  • L-Fucitol is the raw material for producing L-fuculose, and is easily obtained from D-galactose, which is inexpensive. Therefore, the method for producing L-fucose of the present invention is an industrially excellent method which is easy and simple and can reduce the cost.
  • the step of synthesizing L-fuculose is described above in the section “1. Method for producing L-fuculose”.
  • Method for producing L-fuculose In the method for producing L-fuculose of the present invention, the various embodiments as mentioned above may be employed.
  • L-fucose isomerase is utilized to obtain L-fucose.
  • L-fucose isomerase include those whose sequences have been registered in the database in, for example, the National Center for Biotechnology Information.
  • a microorganism When using L-fucose isomerase, a microorganism may be transformed to express L-fucose isomerase, and the produced L-fucose isomerase may be then isolated for use.
  • the microorganism transformed to express L-fucose isomerase or the treated microbial cell product of the microorganism may be used.
  • the transformant may be made in the same way as the technique described in the aforementioned section “1-3. Method using genetically engineered strain”.
  • a co-expressing transformant is made and used to synthesize L-fucose from L-fucitol in one reaction system.
  • L-fucose is synthesized from L-fucitol in the presence of at least one of the following: 1) a microorganism transformed to express a microorganism-derived protein which is not able to substantially synthesize a ketohexose other than L-fuculose from L-fucitol and which has dehydrogenase activity which results in synthesis of L-fuculose from L-fucitol, and a protein which is able to synthesize L-fucose from L-fuculose; and 2) a treated microbial cell product of the microorganism.
  • the aforementioned protein (A) may be isolated from Gluconobacter oxydans IFO 3255.
  • the protein of the present invention includes a protein which is substantially identical to (A), such as the protein described in (B).
  • the substantially identical protein may also be isolated from the bacterial strains described in Table 1.
  • the protein of the present invention specified by the aforementioned sequence is not limited by the source from which the protein has been isolated.
  • the aforementioned protein may be produced by preparing a transformed microorganism based on the aforementioned sequence using gene recombinant technology.
  • “several” varies depending on a position and a type of the amino acid in the three dimensional structure of the protein, and wherein such change or changes do not have a signficant negative effect on the three dimensional structure and the activity of the protein. Specifically, “several” ranges from 1 to 100, preferably from 1 to 50, more preferably from 1 to 30, and still more preferably from 1 to 10. However, in the case of the protein (B), it is desirable that the protein (B) retains the enzyme activity at about a half or more, preferably 80% or more, more preferably 90% or more, and particularly preferably 95% or more of the protein (A) under the conditions of 30° C. and pH 9.5.
  • the amino acid mutations described in (B) may be obtained by modifying the nucleotide sequence of the gene encoding the protein of the present invention using, e.g., site-directed mutagenesis so that the amino acid at specific position is substituted, deleted, inserted, or added.
  • the polypeptide having the modified nucleotide sequence may be obtained by conventionally known mutagenesis treatments, including in vitro treatment of the DNA encoding (A) with hydroxylamine and treating Escherichia coli having the DNA encoding (A) with ultraviolet ray irradiation or a mutagen such as N-methyl-N′-nitro-N-nitrosoguanidine (NTG) or nitrous acid, which are typically used in artificial mutagenesis.
  • the aforementioned mutations may also include naturally occurring mutations, such as differences depending on species and bacterial strains of the microorganisms.
  • the DNA molecule encoding the protein substantially identical to the protein described in SEQ ID NO:16 may be obtained by expressing the DNA molecule having the aforementioned mutations in the appropriate cell and examining the present enzyme activity of an expressed product.
  • a polynucleotide encoding the protein for the method for producing L-fuculose may include a polynucleotide encoding the amino acid sequence described in SEQ ID NO:16. There can be a plurality of nucleotide sequences which define one amino acid sequence due to codon degeneracy. That is, the polynucleotide of the present invention includes the polynucleotides having the nucleotide sequences encoding the following proteins:
  • the nucleotide sequence encoding the amino acid sequence described in SEQ ID NO:16 may include the nucleotide sequence of SEQ ID NO:15.
  • the polynucleotide which is substantially identical to the polynucleotide of SEQ ID NO:15 may be obtained by isolating a polynucleotide which hybridizes with the nucleotide sequence which is complementary to the nucleotide sequence of SEQ ID NO:15, or a probe prepared from the nucleotide sequence under stringent conditions and encodes the protein which has dehydrogenase activity which results in synthesis of L-fuculose from L-fucitol.
  • the preferable polynucleotides of the present invention may include the following:
  • the probe may be prepared based on the nucleotide sequence of SEQ ID NO:15 by a conventional method. Using the probe, the polynucleotide which hybridizes with this probe may be isolated by conventional method.
  • the DNA probe may be prepared by amplifying the nucleotide sequence cloned into the plasmid or the phage vector, cutting out the desired nucleotide sequence using restriction enzymes, and extracting it. Restriction sites may be chosen depending on the DNA.
  • the “stringent conditions” refers to when a so-called specific hybrid is formed and a non-specific hybrid is not formed. Examples thereof may include when a pair of DNA sequences with high homology, e.g., DNA sequences having the homology of 50% or more, more preferably 80% or more, still more preferably 90% or more, and particularly preferably 95% or more, hybridize to each other, whereas DNAs with lower homology do not hybridize, and with washing conditions typical of an ordinary Southern hybridization, i.e., hybridization at salt concentrations equivalent to 1 ⁇ SSC and 0.1% SDS at 60° C., and preferably 0.1 ⁇ SSC and 0.1% SDS at 60° C.
  • the genes which hybridize under these conditions may include those in which a stop codon is present, or an active center has been lost due to mutation. However, these features may be easily removed by ligating the gene to the commercially available vector, expressing it in the appropriate host, and measuring the enzyme activity of the expressed product by the methods described in the following Example.
  • the polynucleotide encoding the aforementioned protein (B), and the polynucleotide (b) it is desirable that the polynucleotide encodes the protein which retains the dehydrogenase activity which results in synthesis of L-fuculose from L-fucitol at about a half or more, more preferably 80% or more, and still more preferably 90% as compared to the protein having the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:1 under the conditions of 30° C. and pH 9.5.
  • the DNA having the nucleotide sequence of SEQ ID NO:15 may be obtained from the chromosomal DNA or the DNA library of Gluconobacter oxydans IFO 3255 or IFO 3171 by PCR (see Polymerase chain reaction, White, T. J. et al; Trends Genet., 5, 185(1989)) or hybridization.
  • Primers used for PCR may be designed based on an internal amino acid sequence determined based on the purified protein having the peptide-synthesizing activity.
  • the full length coding region of the present protein may be amplified using primers corresponding to the 5′ non-translated region and 3′ non-translated region in PCR.
  • the primer may be synthesized using a DNA synthesizer model 380B (Applied Biosystems) and a phosphoamidite method (see Tetrahedron Letters (1981), 22, 1859) in accordance with conventional methods.
  • the PCR reaction may be performed by using Gene Amp PCR System 9600 (PERKIN ELMER) and TaKaRa LA PCR in vitro Cloning Kit (Takara Bio Inc.) in accordance with the instructions supplied by the the manufacturer.
  • one preferable embodiment of the method for producing L-fuculose may be when NADH oxidase is added to the reaction system.
  • NADH oxidase used herein:
  • the aforementioned protein (C) may be isolated from Gluconobacter oxydans IFO 3255.
  • the protein of the present invention includes a protein which is substantially identical to (C), such as the protein described in (D). Furthermore, the substantially identical protein may be isolated from the bacterial strains described in Table 1.
  • the polynucleotide encoding the protein for the method for producing L-fuculose of the present invention includes that shown in SEQ ID NO: 18. There can be a plurality of nucleotide sequences which define one amino acid sequence because of codon degeneracy. That is, the polynucleotide of the present invention includes polynucleotides having the nucleotide sequences encoding the following proteins:
  • the preferable polynucleotides of the present invention may include the following polynucleotides:
  • the polynucleotide encoding NADH oxidase is incorporated into a recombinant polynucleotide such as an expression vector
  • the aforementioned polynucleotide alone may be incorporated, or it may be incorporated with a polynucleotide encoding the protein which has dehydrogenase activity which results in synthesis of L-fuculose from L-fucitol.
  • the protein with NADH oxidase activity and the protein with dehydrogenase activity may be expressed in separate transformants. Alternatively, they may be co-expressed in the same microorganism.
  • NADH oxidase when NADH oxidase is used with another dehydrogenase in the NAD reproduction system, the dehydrogenase, the NADH oxidase, and NAD must be present in the same reaction system.
  • the dehydrogenase and the NADH oxidase may be produced in separate hosts.
  • the untreated microbial cells are used as a catalyst, it is desirable to co-express both enzymes in the same host.
  • Each of the acetobacterium strains was cultured on YPG agar medium (10 g/l glycerol, 0.3 g/l yeast extract, 0.3 g/l peptone, 20 g/l agar, pH 6.5) at 30° C. for 18 to 66 hours, and one platinum loopful of each culture was then seeded into 0.5 mL of a liquid medium containing 10 g/l (61 mM) L-fucitol, 10 g/l glycerol, 3 g/l yeast extract, 3 g/l peptone, and 20 g/l CaCO 3 pH 6.5 that had been sterilized by autoclaving at 120° C. for 20 minutes.
  • HPLC high performance liquid chromatography
  • L-fucose, L-fucitol, and L-fuculose eluted at about 7.6, 11.9 and 13.6 minutes, respectively.
  • Commercially available preparations of L-fucitol and L-fucose supplied from Sigma were used as standards to determine their eluted positions and their concentrations for comparison.
  • L-fuculose no commercially available product was available for use as a standard.
  • a substance produced from L-fucose using L-fucose isomerase which will be described below in detail, was used as L-fuculose.
  • the sum of the integral value of the peak (peak area value) of remaining fucose and the integral value of the peak of produced L-fuculose on a chart was scarcely changed and was constant regardless of before and after reacting with L-fucose isomerase.
  • the peak area value of L-fuculose per unit concentration was regarded to be the same as that of L-fucose, and the concentration of L-fuculose was calculated accordingly in the preparation.
  • the detection limit is about 1 mM in the analyses in the Examples. In many analyses, an unidentified peak appeared which seemed to be a transformed product derived from L-fucitol because of its peak area size. This peak was observed at about 8.0 minutes.
  • BP concentration of this unidentified substance
  • Gluconobacter and “ Acetobacter ” are sometimes abbreviated as “G.” and “A.”, respectively.
  • each of the acetobacterium strains was cultured on YP-dulcitol agar medium (10 g/l dulcitol, 0.3 g/l yeast extract, 0.3 g/l peptone, 20 g/l agar, pH 6.5) at 30° C. for 18 to 66 hours, and one loopful of each strain was then seeded into 0.5 mL of the liquid medium containing 10 g/l L-fucitol, 3 g/l yeast extract and 3 g/l peptone pH 6.5 that had been sterilized by autoclaving at 120° C. for 20 minutes. Cultivation was then performed with shaking at 30° C. for 42 hours. This cultured medium was analyzed in the same way as in Example 1. The analysis results are shown in Table 2.
  • L-Fucose isomerase (EC 5.3.1.3., referred to hereinbelow as “FucI”) catalyzes isomerization between L-fuculose and L-fucose. If the compound assumed to be L-fuculose in Examples 1 and 2 is L-fuculose, this compound should convert to L-fucose in the presence of FucI.
  • L-fucose e.g., Sigma
  • this product is possible to perform HPLC analysis or a colorimetric quantification using L-fucose dehydrogenase described in Patent No. 3132913 Publication.
  • FucI was isolated from E. coli K12 strain. This enzyme has been registered with accession number AAC75844 in the database of National Center for Biotechnology Information. To express this enzyme in large amounts in E. coli , and also to insert a histidine tag at the N terminus to facilitate purification, pQE30 (QIAGEN) was used as the expression vector for FucI.
  • PCR primers 1 and 2 shown as SEQ ID NO:1 and SEQ ID NO:2, respectively, were prepared. PCR was performed using a template of genomic DNA from E.
  • coli W3100 that had been obtained by the standard method to yield a fragment with a length of about 1.8 kbp.
  • This fragment and the pQE30 vector were digested with SphI and HindIII and purified, and then ligated to produce plasmid pQE30FucIH6 (hereinbelow referred to as FucIH6).
  • the expressed protein contained the His-tag sequence at the N terminus.
  • the entire nucleotide sequence and the deduced amino acid sequence of the expressed fucIH6 are shown in SEQ ID NOS:3 and 4, respectively.
  • E. coli JM109 strain was transformed with this plasmid, resulting in FucIH6-expressing strain E. coli JM109/pQE30FucIH6.
  • This bacterial strain was cultured at 37° C. in LB medium, and the expression of fucIH6 was induced by adding IPTG to a final concentration of 1 mM when the absorbance at 600 nm was about 0.4. The cultivation was continued for 2 hours after this induction. Then, the microbial cells were collected by centrifuging the medium, and were washed with 50 mM Tris-HCl (pH 8.0). The washed microbial cells were disrupted by sonication at 200W for 10 minutes. The disrupted product was then centrifuged to obtain a supernatant which was used as a crude enzyme solution.
  • Imidazole to a final concentration of 10 mM and NaCl to a final concentration of 0.3 M were added to this crude enzyme solution, which was then mixed with 1 ml of Ni-NTA resin (QIAGEN) which had been equilibrated with 50 mM Tris-HCl (pH 8.0), 10 mM imidazole, and 0.3 M NaCl (hereinafter, “buffer W”).
  • Buffer W 50 mM Tris-HCl (pH 8.0), 10 mM imidazole, and 0.3 M NaCl
  • the eluted fraction thus obtained was dialyzed against 50 mM Tris-HCl (pH 8.0), 1 mM 2-mercaptoethanol, and 0.1 mM MnCl 2 , and further concentrated through a membrane as needed, for subsequent experimentation.
  • the resulting enzyme solution was subjected to SDS-PAGE. As a result, an almost single band with a molecular weight of about 67 kDa, which seemed to be FucIH6, was observed.
  • the bacterial strains listed in Table 3 were used for the conversion of L-fucitol by acetobacterium .
  • One platinum loopful of each refreshed acetobacterium strain was seeded into 0.5 mL of a liquid medium of 10 g/l L-fucitol, 10 g/l glycerol, 3 g/l yeast extract, and 3 g/l peptone at pH 6.5, and cultured with shaking at 30° C. for 42 hours.
  • the medium from which glycerol had been removed was used for only the cultivation of Gluconobacter xylinus subsp. xylinus ATCC 53582 strain.
  • the 96-well microplate with 2 ml per well was used for the cultivation.
  • the resulting cultured liquid was centrifuged to remove the microbial cells, and an equivalent volume of 0.2 M potassium phosphate buffer (pH 7.0) containing 2 mM 2-mercaptoethanol, 0.2 mM MnCl 2 and 0.4 mg/ml FucIH6 (each final concentration is a half thereof) were added to react at 37° C. for 2 hours. At that time, an experimental group without FucIH6 was made and used as the control.
  • L-fucose was also quantified by the calorimetric method using L-fucose dehydrogenase.
  • the quantification was performed by adding L-fucose dehydrogenase (Kikkoman Corporation, NADP-dependent) to the final concentration of 0.2 mg/ml, NADP to the final concentration of 1 mM in 0.1 M glycine-NaOH buffer (pH 9.5) to the appropriately diluted FucIH6 reaction solution to react at 37° C. for 30 minutes.
  • the changes in the absorbance at 340 nm induced by NADPH production were measured. The results are shown in Table 3.
  • Example 3 it was demonstrated that L-fuculose can be produced from L-fucitol by acetobacterium , and L-fuculose can be converted into L-fucose using FucI.
  • L-fucose using L-fucitol it is possible to separately produce L-fuculose from L-fucitol and L-fucose from L-fuculose as in Example 3.
  • acetobacterium itself could convert L-fuculose to L-fucose, it would become possible to advantagously produce L-fucose from L-fucitol using a single bacterial strain.
  • G. oxydans IFO 3171 strain a typical strain which is able to synthesize L-fuculose from L-fucitol, was transformed to construct a strain which expresses FucI isolated from E. coli .
  • the expression system of the specific protein in the acetobacterium it is possible to utilize the method of using the plasmid pSA19 described in Biosci. Biotechnol. Biochem., 67: 584-591 (2003).
  • coli was obtained by PCR as in Example 3, and this was inserted into the multicloning sites of pSA19 to yield a FucI expression plasmid, named pSA19FucI.
  • pSA19FucI a FucI expression plasmid
  • no His-tag was added to FucI, so the wild-type FucI was expressed.
  • Primers having the sequences of SEQ ID NOs:5 and 6 were used in PCR to clone from E. coli . This means that the predicted amino acid sequence of the expressed FucI and the fucI gene inserted into pSA19FucI are identical to the wild-type enzyme.
  • G. oxydans IFO 3171 strain was transformed with pSA19FucI using the method of the aforementioned reference.
  • one platinum loopful thereof was seeded into 3 ml of the liquid medium containing 10 g/l L-fucitol, 10 g/l glycerol, 3 g/l yeast extract, and 3 g/l peptone pH 6.5, and cultured with shaking at 30° C. for 18 hours.
  • Microbial cells were collected by centrifuging 1 ml of the medium, and then washed with 0.1 M Tris-HCl buffer (pH 8.0). The washed microbial cells were resuspended in 0.5 ml of the same buffer.
  • L-fuculose which was synthesized by the conversion with the wild-type strain was further converted into L-fucose in the FucI-expressing strain. This enabled production of L-fucose from L-fucitol with the acetobacterium strain alone.
  • G. oxydans IFO 3255 strain G. oxydans IFO 3171 strain, G. roseus AJ 2840 strain, and A. turbidans AJ 2908 strain were used in the experiments.
  • One platinum loopful of the bacterial strain that had been refreshed on YPG agar medium was seeded into a sterilized liquid medium containing 10 g/l glycerol, 10 g/l L-fucitol, 0.3 g/l yeast extract, 0.3 g/l peptone, and 20 g/l CaCO 3 pH 6.5, and cultured in a test tube at 30° C. for 42 hours. Subsequently, 1 ml of the cultured medium was dispensed.
  • the microbial cells were collected by centrifugation, and then washed with 0.1M potassium phosphate buffer (pH 6.0) or 0.1 M Tris-HCl buffer (pH 8.0) or 0.1 M glycine NaOH buffer (pH 8.8).
  • the washed microbial cells were resuspended in 0.5 ml of each buffer.
  • L-fucitol was added to a final concentration of 10 g/l, and the mixture was reacted at 30° C. for 66 hours in the 96-well microplate having 2 ml volume per well.
  • the microbial cells were removed by centrifugation, and the supernatant was subjected to HPLC analysis. The results are shown in FIG. 3 . In the figure, NT indicates that the experiment was not performed.
  • G. oxydans IFO 3255 strain was used in the experiments.
  • the cultured liquid was obtained in the same way as in Example 5 (except that CaCO 3 was not added to the cultivation medium), and 1 ml of the cultured liquid was dispensed.
  • the microbial cells were collected by centrifugation and washed with 0.1 M potassium phosphate buffer (pH 6.0). The washed microbial cells were resuspended in 0.5 ml of the same buffer. L-fucitol to a final concentration of 10 g/l and EDTA or MgCl 2 to a final concentration of 10 mM were added thereto, and the mixture was reacted at 30° C. for 90 hours in the 96-well microplate having 2 ml volume per well. After the reaction, the microbial cells were removed by centrifugation, and the supernatant was subjected to HPLC analysis. The results are shown in FIG. 4 .
  • D-Arabitol dehydrogenase present on the cell membrane has also been referred to as glycerol dehydrogenase or D-sorbitol dehydrogenase because of the broadness of its substrate specificity.
  • this enzyme has been referred to as D-sorbitol dehydrogenase, and its gene has been cloned and its nucleotide sequence has been determined.
  • the gene encoding a D-arabitol dehydrogenase catalytic subunit is hereinbelow designated as sldA
  • the protein encoded by sldA is hereinbelow designated as SldA.
  • a DNA fragment which lacks an internal partial sequence of sldA was prepared. This fragment was introduced into an acetobacterium to induce homologous recombination of sldA on the chromosomal DNA. To detect the mutant strain, a kanamycin resistant gene Kmr was introduced with the defective sldA fragment. Thus, the mutant strain was easily detected by growing on a medium containing kanamycin.
  • a fragment containing an internal partial sequence of sldA was obtained by PCR using the primers shown in SEQ ID NOS:7 and 8 (having KpnI and PstI recognition sites, respectively).
  • genomic DNA prepared from Gluconobacter oxydans IFO 3255 strain whose sldA nucleotide sequence had been reported was used.
  • genomic DNA prepared from Gluconobacter oxydans IFO 3171 strain was used, the fragment having the anticipated length was also amplified. This result was due to the high homology of the sequence.
  • the Kmr gene was obtained by PCR using the template plasmid pHSG298 (Takara Bio Inc.) bearing Kmr, and the primers shown in SEQ ID NOS:9 and 10.
  • the fragment obtained by PCR with the template of the acetobacterium genomic DNA was digested with KpnI and PstI and purified, and then subcloned into pUC18 (Takara Bio Inc.) that had been digested with KpnI and PstI and purified.
  • E. coli JM 109 strain was transformed with this plasmid, and cultured. Subsequently, the plasmid was extracted, purified, and digested with BamHI and BglII and purified.
  • the fragment prepared by PCR with pHSG298 containing Kmr was ligated thereto to yield a novel plasmid. E. coli JM109 strain was transformed with this plasmid, and cultured.
  • the plasmid was extracted, purified, further digested with KpnI and PstI, and purified to yield a sldA fragment with the Kmr gene inserted therein.
  • Each of these fragments derived from IFO 3255 strain and IFO 3171 strain was introduced into IFO 3255 strain and IFO 3171 strain, respectively, by electroporation (electrode interval: 0.5 mm, 14.0 kV/cm).
  • the strains capable of growing on a kanamycin-containing plate were selected to yield G. oxydans IFO 3255 sldA:Kmr strain and G. oxydans IFO 3171 sldA:Kmr strain.
  • L-fucitol conversion was performed. 1 mL of the cultured medium obtained as in Example 6 was dispensed. The microbial cells were collected by centrifugation and washed with 0.1 M glycine NaOH buffer (pH 8.8). Then, the cells were resuspended in 0.5 ml of the same buffer. L-fucitol to a final concentration of 10 g/l and MgCl 2 to a final concentration of 10 mM were added thereto, and the mixture was reacted at 30° C. for 66 hours in the 96-well microplate having 2 ml volume per well.
  • Example 7 Since it was demonstrated in Example 7 that an enzyme other than SldA which synthesizes L-fuculose from L-fucitol was present, a search for this enzyme activity was conducted.
  • a candidate of the enzyme of interest seemed to be NAD(P)-dependent dehydrogenase because synthesis of L-fuculose from L-fucitol involves oxidation. It has been reported that D-arabitol dehydrogenase derived from red algae Galdieria sulphuraria catalyzes the oxidation of L-fucitol in an NAD-dependent manner (Planta 202: 487-493 (2003)). However, this activity is weak, and no product has been identified. Thus, whether L-fuculose is formed or a product other than L-fuculose is produced as in the case of SldA has not been examined.
  • the microbial cells were collected by centrifuging this medium, washed with 9 g/l of NaCl, and sonicated with 200 W for 10 minutes to yield the disrupted microbial cells. These cells were further centrifuged at 15,000 g for 15 minutes to yield the supernatant, which was then used as a cell-free extract. Using this cell-free extract as an enzyme source, L-fucitol and NAD(P) at final concentrations of 10 mM and 1 mM, respectively, were reacted in the presence of 0.1 M glycine NaOH buffer (pH 8.8) or 0.1 M potassium phosphate buffer (pH 6.0) at 30° C., to measure the L-fucitol oxdation activity.
  • the experiment was also performed without L-fucitol as the control.
  • the oxidation activity was quantified by the increase in absorbance at 340 nm caused by the production of NAD(P)H.
  • One activity unit was defined as the activity to oxidize 1 ⁇ mol of the substrate in one minute. The results are shown in Table 4.
  • the cell-free extract obtained in Example 8 was added to 0, 0.8, 1.6, and 4.0 mg/ml as the total protein final concentrations to the reaction solution, and left to stand with 10 g/l L-fucitol, 0.1M glycine NaOH (pH 8.8), 0.03 mM FAD, 1 mg/ml BSA, and 2 mM NAD at 30° C. for 90 hours. Subsequently, the product was subjected to HPLC analysis. In order to identify NAD dependency of the L-fucitol conversion, a control without added NAD, and a control with added NADH oxidase for the purpose of supplying NAD were used.
  • NADH oxidase caused synthesis of NAD and hydrogen peroxide from NADH and oxygen (Nacalai Tesque, derived from Bacillus liqueniformis ). NADH produced during the reaction would most likely be reconverted into NAD by NADH oxidase, and the NAD would then be reused for the oxidation of L-fucitol. NADH oxidase was added to a final concentration of 0.1 U/ml. Catalase to a final concentration of 10 U/ml was also added for the purpose of consuming the hydrogen peroxide. The results are shown in Table 5.
  • NADH oxidase activity (NADH oxidation) was present in the acetobacterium cell-free extract, the detection of this enzyme was attempted.
  • the cell-free extract obtained in Example 8 was reacted with NADH and FAD at final concentrations of 0.2 mM and +0.03 mM, respectively, in the presence of 0.1M Tris-HCl buffer (pH 8.0) or 0.1M glycine NaOH buffer (pH 8.8) at 30° C., to measure the NADH oxidation activity.
  • the group without the extract was used as a control.
  • the oxidation activity was quantified by measuring the decrease in absorbance at 340 nm caused by the conversion of NADH to NAD.
  • One activity unit was defined as the activity to oxidize 1 ⁇ mol of NADH in one minute at 30° C. The results are shown in Table 6.
  • NADH oxidation activity was detected in both buffers at pH 8.0 and 8.8.
  • pH 8.0 the addition of FAD facilitated this activity.
  • the activity of many known NADH oxidases has been reported to be FAD-dependent.
  • the NADH oxidation activity present in the extract used in this experiment might be due to a similar NADH oxidase.
  • Bacterial strain Gluconobacter oxydans IFO 3255 or IFO 3171 strain
  • the bacterial strain was refreshed by culturing on YPG medium (3 g/l peptone, 3 g/l yeast extract, 1 g/l glycerol, pH 6.5) containing 20 g/l agar at 30° C. for two nights. These refreshed microbial cells were inoculated into a test tube containing 3 ml of autoclaved medium, and cultured with shaking at 30° C. for 24 or 66 hours. After the cultivation, the microbial cells were collected by centrifuging 3 ml of the cultured liquid, washed with 25 mM Tris-HCl buffer (pH 8.0), and resuspended in 0.3 ml of the same buffer. This was then subjected to sonication to disrupt the microbial cells, and then centrifuged at 200,000 g for 30 minutes to yield the supernatant. This was used as a sample to measure enzyme activity.
  • YPG medium 3 g/l peptone, 3 g/l yeast extract, 1
  • the enzyme activity of FcDH was measured by appropriately adding the enzyme solution to 0.2 M Glycine NaOH (pH 9.5), 1 mM MgCl 2 , 10 mM L-fucitol, and 1 mM NAD, and reacting the mixture at 30° C.
  • the activity was quantified by monitoring the change in absorbance at 340 nm to detect the production of NADH from NAD caused by oxidation. The measurement was started when NAD was added. The change in absorbance in the first minute was taken as the initial rate, and the activity was calculated therefrom. The group without L-fucitol was used as a blank.
  • the FcDH activity the activity to oxidize 1 ⁇ mol of L-fucitol in one minute at 30° C.
  • Results The measurement results are shown in Table 7. Using both the FcDH specific activity per unit protein weight and the activity quantity obtained from the unit amount of the cultured liquid as indicators, G. oxydans IFO 3255 was used as the FcDH-producing bacterial strain in the following medium: 10 g/l D-mannitol, 5 g/l yeast extract, 5 g/l peptone (pH 6.5), for 24 hours.
  • oxydans IFO 3255 XYLITOL 66 5.0 10.5 52.4 G. oxydans IFO 3255 XYLITOL 24 7.2 13.9 100.2 G. oxydans IFO 3255 GLYCEROL 66 3.8 9.4 35.9 G. oxydans IFO 3171 GLYCEROL 24 5.4 5.7 31.1 G. oxydans IFO 3171 D-MANNITOL 66 5.0 0.4 2.0 G. oxydans IFO 3171 D-MANNITOL 24 7.6 2.7 20.4 G. oxydans IFO 3171 D-MANNITOL 66 7.5 1.9 14.0 G. oxydans IFO 3171 D-ARABITOL 24 9.2 1.8 16.0 G.
  • the stored G. oxydans IFO 3255 strain was refreshed by culturing in the YPG agar medium at 30° C. for two nights. These refreshed microbial cells were inoculated into the test tube containing 3 ml of autoclaved medium, and cultured with shaking at 30° C. for 18 hours. This was seeded at 1% (v/v) in the 500 ml Sakaguchi flask containing 100 ml of the same autoclaved medium, and cultured at 30° C. for 24 hours. After the cultivation, the microbial cells were collected by centrifugation, washed with 25 mM Tris-HCl buffer (pH 8.0), and suspended in the same buffer at 1/25 the volume of the cultured medium.
  • the fraction containing the FcDH activity was collected, concentrated, dialyzed against 50 mM potassium phosphate buffer (pH 6.0), and applied to Q-Sepharose 16/10 (Amersham Biosciences) that had been equilibrated with the same buffer.
  • the protein which absorbed onto the carrier was eluted by increasing the NaCl concentration in the buffer from 0 M to 0.5 M.
  • the FcDH activity was detected in the fraction having a NaCl concentration of about 0.4 M.
  • the fraction containing the FcDH activity was collected, concentrated, and then applied onto Superdex 200 16/60 (Amersham Biosciences) that had been equilibrated with 50 mM potassium phosphate buffer (pH 6.0).
  • NADH Oxidase Purification of NADH Oxidase (Hereinafter “NOX”)
  • the stored G. oxydans IFO 3255 strain was refreshed by culturing in the YPG agar medium at 30° C. for two nights. These refreshed microbial cells were inoculated into the test tube containing 3 ml of autoclaved medium, and cultured with shaking at 30° C. for 18 hours. This was seeded at 1% (v/v) in a 500 ml Sakaguchi flask containing 100 ml of the same autoclaved medium, and cultured at 30° C. for 24 hours. After the cultivation, the microbial cells were collected by centrifugation, washed with 25 mM Tris-HCl (pH 8.0), and suspended in the same buffer at 1/25 the volume of the cultured medium.
  • the fraction containing the NOX activity was collected, concentrated, dialyzed against the following buffer (50 mM Tris-HCl, pH 8.0, 0.6 M (NH 4 ) 2 SO 4 ), and applied onto Phenyl Sepharose 16/10 that had been equilibrated with the same buffer.
  • the protein which absorbed onto the carrier was eluted by decreasing the (NH 4 ) 2 SO 4 concentration in the buffer from 0.6 M to 0 M.
  • the NOX activity was detected in the fraction having a (NH 4 ) 2 SO 4 (NH 4 ) 2 SO 4 concentration of about 0.2 M.
  • the fraction containing NOX activity was collected, concentrated, dialyzed against 50 mM Tris-HCl (pH 8.0), then concentrated, and subsequently applied to 1.5 ml of a FAD-agarose resin (Sigma) that had been equilibrated with the same buffer.
  • the unabsorbed proteins were thoroughly washed with the same buffer.
  • the absorbed protein was then eluted with a buffer of 50 mM Tris-HCl and 1 mM FAD.
  • NOX was absorbed specifically to the resin, and subsequently eluted by the addition of the buffer containing FAD. This eluted fraction was collected, and concentrated. Its purity was then examined on SDS-PAGE. As a result, NOX was observed as a single band whose molecular weight was estimated to be about 15 kDa ( FIG. 7 ).
  • the purified NOX solution was applied to Superdex 200 16/60 that had been equilibrated with 50 mM Tris-HCl (pH 8.0). Consequently, NOX was eluted at a position at which the molecular weight was estimated to be 34 kDa.
  • the sample obtained by purifying NOX as a single band on SDS-PAGE and eluting through Superdex 200 16/60 was used as a purified enzyme preparation.
  • the NOX activity was measured in accordance with the following standard procedure.
  • the enzyme was appropriately added to 0.1M Tris-HCl (pH 8.0), 30 ⁇ M FAD, 5 mM EDTA, 2 mM DTT, 0.2 mM NADH, and reacted at 30° C.
  • the activity was quantified by monitoring reduction in absorbance at 340 nm caused by the production of NAD from NADH.
  • the measurement was started at the addition of NADH.
  • the change in absorbance in the first minute was taken as an initial rate, and the activity was calculated therefrom.
  • the experimental group without the enzyme source was used as a blank.
  • the NOX activity the activity to oxidize 1 ⁇ mol NADH in one minute at 30° C. was defined as one unit (U). The measurement was taken three consecutive times. Their mean value was taken and is shown in the figure with the standard deviation.
  • Example 12 Using the FcDH preparation purified in Example 12 as the enzyme source, the protein was characterized.
  • the reaction was performed in a solution containing 0.1M pH buffer, 1 mM MgCl 2 , 10 mM L-fucitol, 1 mM NAD, and 1.1 ⁇ g/ml purified FcDH at 30° C., and the production of NADH was determined from the measurement of A 340 .
  • pH buffers sodium acetate buffer (pH 4.8, 5.7), potassium phosphate buffer (pH 6.4, 7.2), Tris-HCl buffer (pH 6.7, 7.6, 8.5), CAPS-NaOH buffer (Sodium cyclohexylaminopropanesulfonate) (pH 8.3, 9.5) and sodium carbonate buffer (pH 10.1, 10.7, 11.2) were used.
  • the specific activity of FcDH measured at each pH ( FIG. 8 ) is reported as the relative percentage as compared to the specific activity at pH 9.5 (maximum specific activity).
  • a solution containing 0.1M pH buffer and 80 ⁇ g/ml purified FcDH was left to stand on ice for 30 minutes, and subsequently the solution was diluted to a final FcDH concentration of 1.6 ⁇ g/ml.
  • the reaction was performed under standard conditions, and the remaining FcDH activity was measured.
  • As the pH buffers sodium citrate buffer (pH 2.6, 3.7, 5.1), sodium acetate buffer (pH 4.7, 5.8), potassium phosphate buffer (pH 6.1, 7.2), Tris-HCl buffer (pH 7.0, 7.8, 8.8), CAPS-NaOH buffer (pH 8.8, 9.5) and sodium carbonate buffer (pH 10.1, 10.6, 11.2) were used.
  • the remaining activity of FcDH was measured at each pH ( FIG. 9 ), and is reported as the relative percentage as compared to the remaining activity at pH 7.2 (maximum stability). As a result, 80% or more remaining activity was observed at pH of about 5 to 10.
  • a solution containing Gly-NaOH (pH 9.5), MgCl 2 , fucitol, and purified FcDH was preincubated at each temperature (25, 30, 37, 45, 55, or 65° C.) for 3 minutes, and then NAD that had been separately preincubated at each temperature was added thereto to start the reaction.
  • the final concentrations in the reaction solution were adjusted to the standard conditions for activity measurement, and the final concentration of FcDH was 1.6 ⁇ g/ml.
  • the specific activity of FcDH measured at each temperature ( FIG. 10 ) is reported as the relative percentage as compared to the specific activity at 37° C. (maximum specific activity).
  • a solution containing Gly-NaOH (pH 9.5) and 80 ⁇ g/ml purified FcDH was left to stand at 0, 25, 30, 37, 50 or 60° C. for 30 minutes.
  • the solution was then diluted so that the final concentration of FcDH was 1.6 ⁇ g/ml.
  • the reaction was performed under standard conditions, and the remaining FcDH activity was measured.
  • the remaining activity of FcDH measured at each temperature is reported ( FIG. 11 ) as the relative percentage as compared to the remaining activity when left to stand at 0° C. Up to 30° C., 80% or more remaining activity was observed.
  • the results of measuring the specific activity of the purified FcDH are shown in FIG. 12 .
  • D-arabitol and L-fucitol reaction inhibition by the substrate was observed at a high concentration of the substrate.
  • a reduction of the specific activity was observed at 5 mM or more of D-arabitol (A) and at 100 mM or more of L-fucitol (B).
  • Lineweaver-Burk plots of the results in (A) and (B) are shown in FIG. 12 (C) and (D), respectively.
  • the K m and k cat values were calculated from the range in which substrate inhibition was not observed, i.e., from a straight line represented by a broken line in FIG. 12 (C) and (D).
  • the k cat and K m values were calculated within the range of substrate concentrations at which no inhibitory effect was observed.
  • no inhibitory effect by the substrate was observed up to the substrate concentration of 100 mM.
  • the oxidation product of L-fucitol is L-fuculose, and the oxidation products of the other substrates were examined.
  • the substrates used were D-arabitol, D-mannitol and D-sorbitol.
  • a reaction solution containing 0.5 g/dl of the substrate, 50 mM Gly-NaOH (pH 9.5), 1 mM NAD, 30 mM FAD, 5 mM MgCl 2 , 0.6 U/ml purified FcDH, 0.2 U/ml NOX, and 0.1 mg/ml catalase was reacted at 30° C. for 16 hours, and analyzed by HPLC ( FIG. 13 ).
  • Each specific activity measured is shown in Table 11 as the relative percentage as compared to the specific activity when MnCl 2 was added, or the maximum specific activity.
  • the activation effect on FcDH was almost equally observed for Mn 2+ , Mg 2+ and Ca 2+ ions.
  • Ni 2+ and Co 2+ ions had no effect.
  • Zn 2+ and Cu 2+ ions had an inhibitory effect.
  • the bivalent metal ion was chelated by EDTA, the activity of FcDH nearly disappeared.
  • FcDH uses NAD as a coenzyme
  • the availability of NAD and NADP was measured.
  • the activity was measured by adding NAD or NADP at various concentrations under standard conditions.
  • the K m value for each coenzyme was calculated by drawing a Lineweaver-Burk plot of the results ( FIG. 14 (B)).
  • the K m value for NAD was determined to be 0.20 mM.
  • NADP was used as the coenzyme, no significant activity was observed (data not shown).
  • Example 13 Using the NOX preparation purified in Example 13 as the enzyme source, the protein was characterized.
  • the reaction was performed in a solution containing 0.1M pH buffer, 30 ⁇ M FAD, 5 mM EDTA, 2 mM DTT, 0.2 mM NADH and 0.055 ⁇ g/ml purified NOX at 30° C., and the reduction of NADH was determined from the measurement at A 340 .
  • pH buffers sodium citrate buffer (pH 3.6, 4.7, 5.7), sodium acetate buffer (pH 4.8, 5.7), potassium phosphate buffer (pH 6.4, 7.2), Tris-HCl buffer (pH 6.7, 7.6, 8.5), and CAPS-NaOH buffer (pH 8.3, 9.5) were used.
  • the specific activity of NOX measured at each pH ( FIG. 15 ) is reported as the relative percentage as compared to the specific activity at pH 5.7, or the maximum specific activity.
  • a solution containing 0.1M pH buffer, 30 ⁇ M FAD, 5 mM EDTA, 2 mM DTT, and 100 ⁇ g/ml purified NOX was left to stand on ice for 30 minutes, and subsequently the solution was diluted so that the final concentration of FcDH was 0.055 ⁇ g/ml.
  • the reaction was performed under standard conditions, and the remaining NOX activity was measured.
  • sodium citrate buffer pH 2.6, 3.7, 5.1
  • sodium acetate buffer pH 4.7, 5.8
  • potassium phosphate buffer pH 6.1, 7.2
  • Tris-HCl buffer pH 7.0, 7.8, 8.8
  • CAPS-NaOH buffer pH 8.8, 9.5
  • sodium carbonate buffer pH 10.1, 10.6, 11.2
  • a solution containing Tris-HCl (pH 8.0), FAD, EDTA, DTT, and purified NOX was preincubated at each temperature (25, 30, 37, 45, 55 or 65° C.) for 3 minutes, and then NADH that had been separately preincubated at each temperature was added thereto to start the reaction.
  • the final concentrations in the reaction solution were adjusted to standard conditions of the activity measurement, and the final concentration of NOX was 0.055 ⁇ g/ml.
  • the specific activity of NOX measured at each temperature ( FIG. 17 ) is reported as the relative percentage (remaining activity) as compared to the specific activity at 37° C., or maximum specific activity.
  • the solution containing 0.1M Tris-HCl (pH 8.0), 30 ⁇ M FAD, 5 mM EDTA, 2 mM DTT, and 100 ⁇ g/ml purified NOX was left to stand at 0, 25, 30, 37, 50 or 60° C. for 30 minutes.
  • the solution was then diluted so that the final concentration of NOX was 0.055 ⁇ g/ml.
  • the reaction was performed under standard conditions, and the remaining NOX activity was measured.
  • the remaining activity of NOX measured at each temperature ( FIG. 18 ) is reported as the relative percentage as compared to the remaining activity when left to stand at 0° C. Up to 50° C., 80% or more remaining activity was observed.
  • the K m value for the substrate was calculated by measuring the activity with a variety of substrate concentrations and drawing a Lineweaver-Burk plot of the results. As a result, the K m value for NADH was determined to be 29 ⁇ M ( FIG. 20 ). Meanwhile, when NADPH was used as the substrate, no significant activity was observed (data not shown).
  • DNA primers corresponding thereto were synthesized from the peptide sequences obtained in Example 16. PCR was performed using genomic DNA prepared from G. oxydans IFO 3255 as the template in accordance with standard methods. The resulting fragment was purified and its nucleotide sequence was analyzed by the DNA sequencer. As a result, the DNA sequences encoding the peptide sequences obtained in Example 16 were determined.
  • FcDH For the expression of FcDH, PCR was performed with the genomic DNA from G. oxydans IFO 3255 strain as the template and the synthetic primers shown in SEQ ID NOS:19 and 20, to yield a fragment of 858 bp including the fcdh gene.
  • the resulting PCR product was digested with EcoRI and PstI and inserted into the corresponding position in the plasmid pUC18 (Takara Bio Inc.) to prepare the plasmid pFEX3052 for the expression of FcDH.
  • PCR was performed with the genomic DNA from G. oxydans IFO 3255 strain as the template and the synthetic primers shown in SEQ ID NOS:21 and 22, to yield a fragment of 605 bp including the nox gene.
  • the resulting PCR product was digested with PstI and HindIII, and inserted into the corresponding position in the plasmid pFEX3052 to prepare the plasmid pFNEX4105 for the co-expression of FcDH and NOX.
  • PCR was performed with plasmid pFNEX4105 as the template and the synthetic primers shown in SEQ ID NOS: 19 and 23, to yield a fragment of 1390 bp including a gene fragment in which the fcdh gene and the nox gene had been ligated in tandem.
  • the resulting PCR product was digested with EcoRI and KpnI, and subsequently inserted into the corresponding position in the plasmid pUC18 to prepare pFNEX4502.
  • PCR was performed with the genomic DNA obtained from E. coli W3110 strain and the primers shown in SEQ ID NOS:24 and 25 synthesized based on the fucI gene sequence described in Accession No. NC — 000913, to yield a fragment of 1873 bp including the fucI gene.
  • the resulting PCR product was digested with KpnI and SalI, and subsequently inserted into the corresponding position in the plasmid pFNEX4502 to prepare the plasmid pFNIEX5706 for the co-expression of FcDH, NOX and FucI.
  • the plasmid pIEX11 for the expression of FucI alone was prepared. This was prepared by performing PCR similar to above using the synthetic primers shown in SEQ ID NOS:26 and 27 to yield the fucI gene, digesting the resulting product with EcoRI and PstI, and inserting it into the corresponding position in the plasmid pUC18.
  • Escherichia coli JM 109 strain (supplied from Takara Bio Inc.) was transformed with the constructed plasmids pFEX3052, pFNEX4105, pFNIEX570 or pIEX11 to produce FcDH-expressing strain E. coli /pFEX3052, FcDH and NOX co-expressing strain E. coli /pFNEX4105, FcDH, NOX, and FucI co-expressing strain E. coli /pFNIEX5706 and FucI-expressing strain E. coli /pIEXI11.
  • TB medium (12 g/l trypton, 24 g/l yeast extract, 4 g/l glycerol, 2.3 g/1 KH 2 PO 4 , 12.5 g/l K 2 HPO 4 ) was used instead of LB medium in some cases, and they were not essentially different, except that a larger amount of microbial cells was obtained.
  • the washed microbial cells were resuspended in 25 mM Tris-HCl (pH 8.0), and disrupted by sonication (200 W, 10 minutes). Then the supernatant was obtained by centrifugation at 14,000 g for 15 minutes, and this was used as the cell-free extract in the various experiments.
  • the result of analysis of the cell-free extracts obtained from E. coli /pUC18, E. coli /pIEX11, E. coli /pFEX3052, E. coli /pFNEX4105 and E. coli /pFNIEX5706 on SDS-PAGE is shown in FIG. 21 . FucI was produced with E. coli /pIEX11.
  • FcDH was produced with E. coli /pFEX3052.
  • FcDH and NOX were produced with E. coli /pFNEX4105.
  • FucI, FcDH, and NOX were produced with E. coli /pFNIEX5706. Each band was observed at the position of the corresponding molecular weight.
  • this cell-free extract was further subjected to ultracentrifugation at 200,000 g for 30 minutes, and the resulting supernatant was used.
  • the supernatant obtained by ultracentrifugation of the cell-free extract obtained from E. coli /pFEX3052 was purified.
  • the crude enzyme solution was dialyzed against 50 mM potassium phosphate buffer (pH 6.0), and applied onto Q-Sepharose 16/10 that had been equilibrated with the same buffer.
  • the protein which absorbed onto the carrier was eluted by increasing the NaCl concentration from 0 M to 0.5 M in the buffer.
  • the fraction containing the FcDH activity was collected, concentrated, and then applied onto Superdex 200 16/60 that had been equilibrated with 50 mM potassium phosphate buffer (pH 6.0).
  • the supernatant obtained by ultracentrifugation of the cell-free extract of E. coli /pFNEX4105 was purified.
  • the crude enzyme solution was applied onto FAD-agarose resin that had been equilibrated with 50 mM Tris-HCl (pH 8.0), and unabsorbed proteins were thoroughly washed with the same buffer.
  • the absorbed protein was then eluted with the buffer containing 50 mM Tris-HCl (pH 8.0) and 1 mM FAD. NOX was specifically absorbed onto the resin, and eluted by the addition of the buffer containing FAD.
  • the fraction containing the NOX activity was collected, concentrated, and then applied onto Superdex 200 16/60 that had been equilibrated with 50 mM Tris-HCl (pH 8.0), to collect the fraction containing the NOX activity. This was concentrated, and its purity was then examined on SDS-PAGE. As a result, rNOX was observed as a single band whose molecular weight was determined to be about 15 kDa ( FIG. 23 ).
  • the FucI activity was measured as follows. The enzyme solution was appropriately added to 0.1M L-fucose, 0.1M Tris-HCl (pH 8.0), and 5 mM MgCl 2 , and reacted at 30° C. The activity was quantified by measuring the amount of L-fuculose produced by the isomerization of L-fucose by HPLC. The activity was calculated from the range in which the amount of produced L-fuculose did not exceed 3 mM. As to the FucI activity, the activity to produce L-fuculose by isomerizing 1 ⁇ mol of L-fucose in one minute at 30° C. was defined as 1 U.
  • catalase was added to convert H 2 O 2 into H 2 O.
  • a commercially available preparation Nacalai Tesque, derived from bovine liver
  • As standard reaction conditions a reaction solution containing 62 mM L-fucitol, 0.1M Gly-NaOH (pH 9.5), 2 mM NAD, 30 ⁇ M FAD, 1 mM MgCl 2 , 0.1 U/ml purified FcDH, 0.1 U/ml purified NOX, 0.2 mg/ml purified FucIH6, and 0.1 mg/ml catalase was reacted at 30° C. with shaking while sampling was appropriately performed.
  • L-fucitol, L-fuculose, and L-fucose were quantified by HPLC.
  • the experimental groups and the results after the reaction for 48 hours are shown in Table 13.
  • the conversion time course in each experimental group is shown in FIG. 24 .
  • open circles, solid circles, and open triangles represent the concentrations of L-fucose, L-fuculose, and L-fucitol, respectively.
  • (B) shows the result under standard conditions (62 mM L-fucitol, 0.1M Gly-NaOH (pH 9.5), 2 mM NAD, 30 ⁇ M FAD, 1 mM MgCl 2 , 0.1 U/ml purified FcDH, 0.1 U/ml purified NOX, 0.2 mg/ml purified FucIH6, and 0.1 mg/ml catalase).
  • (A) shows the result without any addition of the enzymes
  • (C) shows the result without addition of FucIH6
  • (D) shows the result without addition of FcDH
  • (E) shows the result without addition of NOX
  • (F) shows the result without addition of catalase.
  • open circles, solid circles, and open triangles represent the concentrations of L-fucose, L-fuculose, and L-fucitol, respectively.
  • A shows the result under standard conditions (120 mM L-fucitol, 0.1M Gly-NaOH (pH 9.5), 2 mM NAD, 30 ⁇ M FAD, 1 mM MgCl 2 , 1 U/ml purified rFcDH, 1 U/ml purified rNOX, 0.2 mg/ml purified FucIH6, and 0.1 mg/ml catalase).
  • B shows the result without addition of NAD
  • C shows the result without addition of FAD
  • D shows the result without addition of catalase
  • E shows the result without addition of FucIH6.
  • the experiment was performed to convert L-fucitol by intact microbial cells without purifying the produced enzyme from the microbial cells.
  • the bacterial strains E. coli /pUC18, E. coli /pIEX11, E. coli /pFEX3052, E. coli /pFNEX4105 and E. coli /pFNIEX5706 were used alone or in combination.
  • E. coli /pFNIEX5706 were used in the reactions having a single type of microbial cells.
  • the experimental groups and the results after 40 hours are shown in Table 15. The conversion time course in each experimental group is shown in FIG. 26 .
  • open circles, solid circles, and open triangles represent the concentrations of L-fucose, L-fuculose, and L-fucitol, respectively.
  • (A) and (D) show the results using E. coli /pUC18
  • B) and (E) show the results using E. coli /pFEX3052
  • C) and (F) show the results using E.
  • H 2 O 2 derived from the NOX activity inhibited the conversion of L-fucitol, and the addition of catalase reversed this inhibition.
  • a catalase gene was further introduced into the FcDH+NOX+FucI co-expressing strain, to obtain a bacterial strain which further co-expresses catalase.
  • KatE protein derived from E. coli was used as catalase.
  • the gene encoding this enzyme has been registered as accession number M55161 in the database in National Center for Biotechnology Information.
  • the following PCR primers SEQ ID NOS:28 and 29 were prepared. PCR was performed with the genomic DNA derived from E.
  • E. coli W3110 strain as the template, to yield a fragment of about 2.5 kbp.
  • the PCR product was digested with SalI and PstI, and introduced into the corresponding position into the plasmid pSTV28 (Takara Bio Inc.) to yield the plasmid pKEX5804 for KatE expression.
  • E. coli /pFNIEX5706 strain was transformed with this plasmid to make the bacterial strain E. coli /FNIC7001, which has both plasmids pFNIEX5706 and pCEX8401.
  • This strain was refreshed by culturing on LB/Amp+Cm (chloramphenicol 0.03 mg/ml) plates at 37° C. The liquid cultivation was performed at 37° C. using the LB/Amp+Cm liquid medium. After inoculating the refreshed microbial cells, IPTG was added to a final concentration of 1 mM. The microbial cells were cultured for 6 to 18 hours, and then collected by centrifugation. The collected microbial cells were washed with 25 mM Tris-HCl buffer (pH 8.0), and used in the subsequent experiments.
  • the TB medium (12 g/l trypton, 24 g/l yeast extract, 4 g/l glycerol, 2.3 g/l KH 2 PO 4 , 12.5 g/l K 2 HPO 4 ) was used instead of the LB medium in some cases, and they were not essentially different except that a larger amount of the microbial cells was obtained.
  • the microbial cells were collected. The washed microbial cells were resuspended in 25 mM Tris-HCl (pH 8.0), disrupted by sonication (200 W, 10 minutes), and centrifuged at 14,000 g for 15 minutes to yield the supernatant. This cell-free extract was used as the enzyme source to measure enzyme activity. E. coli /pFNIEX5706 strain was also used as a control.
  • the conversion experiment of L-fucitol by the washed intact microbial cells of E. coli /FNIC7001 strain cultured at 27, 30, 33, or 37° C. using TB/Amp+Cm was performed.
  • the reaction solution containing 380 mM L-fucitol, 0.2 M Gly-NaOH (pH 9.5), 50 ⁇ M riboflavin, 1 mM MnCl 2 , 0.1 mg/ml Amp, 0.03 mg/ml Cm, and washed microbial cells (adjusted so that the final concentration in the reaction solution was A 610 10.0) was reacted at 30° C. with shaking, while sampling was appropriately performed.
  • L-fucitol, L-fuculose, and L-fucose were quantified by HPLC.
  • the experimental groups and the analysis results after 45 hours are shown in Table 17, and the conversion time course in each experimental group is shown in FIG. 27 .
  • the enzyme activity in the cell-free extract prepared from the microbial cells cultured at each temperature is shown in Table 18.
  • open circles, solid circles, and open triangles represent the concentration of L-fucose, L-fuculose, and L-fucitol, respectively.
  • the microbial cells cultured at 27° C. (FIG. A) 30° C. (FIG. B), 33° C. (FIG. C) or 37° C. (FIG. D) were used for the conversion reaction.
  • the higher the microbial cell culture temperature the higher the enzyme activity per unit protein amount.
  • the conversion rate was faster when culturing at 27, 30 or 33° C. than at 37° C.
  • the present invention is suitable for the industrial production of L-fucose.
  • the present invention is expected to contribute to various fields utilizing L-fucose.

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US20100317068A1 (en) * 2008-01-18 2010-12-16 Shinji Kuroda Method for producing serine derivative and protein used for the same
US20100330622A1 (en) * 2007-12-21 2010-12-30 Sergey Vasilievich Smirnov Bacterium producing a product of a reaction catalyzed by a protein having 2-oxoglutarate-dependent enzyme activity and a method for manufacturing the product
US20110081678A1 (en) * 2008-05-12 2011-04-07 Rie Takeshita Method for producing beta-alanyl-amino acid or derivative thereof
WO2016150629A1 (fr) * 2015-03-26 2016-09-29 Basf Se Production biocatalytique de l-fucose

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JP4452914B2 (ja) * 2003-09-08 2010-04-21 味の素株式会社 新規トランスポータタンパク質
JP4655539B2 (ja) * 2004-08-06 2011-03-23 味の素株式会社 アシラーゼを用いたβアミノ酸の製造方法
JP4806963B2 (ja) 2005-05-20 2011-11-02 味の素株式会社 β−ヒドロキシアミノ酸の製造方法およびこれに用いる酵素
EP1882737B1 (fr) * 2005-05-20 2011-09-14 Ajinomoto Co., Inc. Procédé de fabrication d'un dérivé de l-sérine et enzyme utilisée dans ledit procédé
EP3486326A1 (fr) 2017-11-21 2019-05-22 Jennewein Biotechnologie GmbH Procédé de purification d'acide n-acétylneuraminique à partir d'un bouillon de fermentation

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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100330622A1 (en) * 2007-12-21 2010-12-30 Sergey Vasilievich Smirnov Bacterium producing a product of a reaction catalyzed by a protein having 2-oxoglutarate-dependent enzyme activity and a method for manufacturing the product
US8524476B2 (en) 2007-12-21 2013-09-03 Ajinomoto Co., Inc. Bacterium producing a product of a reaction catalyzed by a protein having 2-oxoglutarate-dependent enzyme activity and a method for manufacturing the product
US20100317068A1 (en) * 2008-01-18 2010-12-16 Shinji Kuroda Method for producing serine derivative and protein used for the same
US8372607B2 (en) 2008-01-18 2013-02-12 Ajinomoto Co., Inc. Method for producing serine derivative and protein used for the same
US20110081678A1 (en) * 2008-05-12 2011-04-07 Rie Takeshita Method for producing beta-alanyl-amino acid or derivative thereof
WO2016150629A1 (fr) * 2015-03-26 2016-09-29 Basf Se Production biocatalytique de l-fucose
CN107454915A (zh) * 2015-03-26 2017-12-08 巴斯夫欧洲公司 L‑岩藻糖的生物催化生产方法
US10428361B2 (en) 2015-03-26 2019-10-01 Basf Se Biocatalytic production of l-fucose
CN107454915B (zh) * 2015-03-26 2022-03-22 巴斯夫欧洲公司 L-岩藻糖的生物催化生产方法

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